Activation of B cells and production of Antibodies Antigen Antibody 1 Antibody 3 Antibody 2

The life history of B lymphocytes Newly produced B cells leave bone marrow and enter circulation Migrate through secondary lymphoid organs and survey for antigens Antigen-recognition; Interaction with helper T cells; clonal expansion; (isotype switching) Low-affinity Plasma cells -> Antibody Germinal Center formation: somatic mutation and affinity maturation High-affinity Plasma cells (->Antibody) and memory B cells (continual) (a few days)(1-2 weeks) (weeks)

(BCR) -> Changes in gene expression include upregulation of B7

T cell clonal expansion; differentiation 'activation' signal but no clonal expansion present Ag T-independent (TI) T-cell dependent (TD) Types of B cell Antigens: T-independent and T-dependent clonal expansion; differentiation BCR Ag T-independent antigens are multivalent (e.g. bacterial polysaccharides or repeating determinants on the surface of viruses) T-dependent antigens must contain a protein component (true of most antigens)

Antigen presentation by B lymphocytes to helper T cells B cells present antigen they are specific for 100,000 times more efficiently than a non-specific antigen

Mechanisms of helper T cell-mediated activation of B lymphocytes

Role of CD40 in B cell activation TCR triggering up- regulates CD40L on T cell CD40 signaling promotes B cell activation, isotype switching CD40 also important in DC, Macrophage function CD40L-deficiency = 'hyper-IgM syndrome' increased expression of cell cycle molecules, survival molecules, promotes isotype switching

Linked help and the Conjugate Vaccine concept Many bacteria are heavily coated with surface polysaccharides Vaccines against these bacteria aim to induce antibodies specific for the polysaccharide e.g. –Haemophilus influenzae Type b vaccine –Pneumococcal vaccine –Meningococcal vaccine But infants and young children mount poor T-independent antibody responses Conjugate vaccines link the polysaccharide to an immunogenic protein carrier so that a T-dependent antibody response can be induced

Mounting a T-dependent antibody response to a polysaccharide in a conjugate vaccine foreign protein sugar (polysaccharide) Polysaccharide Specific B cell T Protein Specific T cell CD40L Cytokines BCR MHC II endosome

Haptens and hypersensitivity reactions Small organic molecules of simple structure do not provoke antibodies by themselves. However, antibodies can be raised against them if the molecule is attached to a protein carrier Termed haptens (from the Greek haptein, to fasten) Some drugs (e.g. Penicillin) can act as haptens and induce antibody-mediated allergic reactions urushiol in poison oak and the nickel in earrings that cause contact dermatitis are both haptens involved in T cell- mediated DTH reactions

Making an antibody response to a hapten proteinhapten Hapten Specific B cell + 1. Hapten covalently attaches to self-protein 2. Hapten specific B cell binds haptenated-protein 3. Complex is internalized and degraded to haptenated peptides 4. Haptenated peptides are presented to T cells 5. B cell receives help and secretes hapten specific antibody CD40L Cytokines T Haptenated- peptide Specific T cell

membrane Ig secretory Ig B cell Plasma Cell After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell After their generation in secondary lymphoid organs, many Plasma Cells home to the bone marrow or mucosal surfaces (or lactating mammary gland) where they live for many months, continually secreting antibody

Production of membrane vs secreted Ig membrane Ig (BCR) C H tm cy polyA secretory Ig (Ab) B cell Plasma Cell V H - B cells express Ig Heavy chain transcripts that include transmembrane and cytoplasmic domains - Plasma cells express Ig Heavy chain transcripts that stop after the C H domains, thereby encoding the same antibody but in a secreted form C H V H

naive B cell activated B cells 3-4 days 12 divisions plasma cells 1 day differentiation 1 day 10 4 Ab/cell/sec antibodies = 4,096 4,096>10 12 B cell antibody response -> clonal replication enters into a higher order upon plasma cell differentation bacteria - possibly dividing every ~60 min 5 days = divisions (Note: the exact numbers are not important)

Ig Heavy chain class (isotype) switching

VDJ    55 kb (cytokines, CD40L) T cell help antigen IgM+ naive B cell IgG+ memory cell IgG secreting plasma cell

Affinity Maturation Affinity maturation occurs in germinal centers (GC) and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells

VHVH VLVL CLCL C H1 CDR 1 23 C H2 C H3 CDR 12 3 Ag Mutations are targeted to antigen binding region of antibody CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen) Ag before Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen after Ag - increasing the binding affinity

Affinity maturation and antibody responses

AID dependent mutator complex DNA replication error ATG... GGC TAT GCT CAC CGT... V C H1 T...GGC, CCT... Met... Gly Tyr Ala His Arg......Gly, Pro... AID = Activation Induced Deaminase (-> deaminates Cytosine on Uracil -> repair proteins then come in and this leads to error prone repair) Somatic mutation of Ig V region in GC B cell -> mutations are actively induced in the V-regions of the antibody heavy and light chain genes Val

ATG... GGC TAT GTT CAC CGT... Met... Gly Tyr Val His Arg... T Val...GGC, CCT......Gly, Pro... V C H1 Somatic mutation of Ig V region in GC B cell -> now encodes antibody molecule with slightly altered antigen binding site -> sometimes, by chance, this site will have an improved ability to bind the inducing antigen (i.e. a higher affinity)

mantle zone GC light zone (bright green staining, FDCs) GC dark zone T zone (red - cell cycle marker-high cells that are rapidly dividing GC B cells) (naive B cells) Germinal Center in Human Tonsil

Germinal Center B B B B B B B B B B B B MØ B T F F B B B B B T T T B B 2. Dark Zone -> GC B cells (blasts) undergo proliferation and somatic mutation of Ig V genes 3. Light Zone -> GC B cells compete to bind antigen displayed on FDC (F) and to receive T cell help -> selection occurs for cells with higher affinity BCR; these cells successfully compete to bind antigen, present peptide- MHC II complexes to T cells and survive -> cells that fail to bind antigen die and are engulfed by macrophages ( MØ) 1.Seeding GC seeded by low affinity B cells that bound antigen and received T cell help 4. Differentiation & Exit -> high affinity (selected) B cells differentiate into long-lived plasma cells and memory B cells that exit the GC MB PC antigen

Memory B cells Generated in germinal centers –therefore we only have strong humoral memory to T-dependent antigens Small, recirculating cells Often isotype switched (e.g. IgG + or IgA + ) Typically have higher affinity for the inducing Ag Longer lived than naïve B cells –Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter

Features of primary and secondary antibody responses